(1) Field of the Invention
The present invention provides a method for manipulating the ratio of various carotenoids in plants as a means for augmenting the accumulation of selected carotenoids. The present invention further relates to transgenic marigold plants which produce various ratios of carotenoids and methods for producing the same. Preferably, various carotenoids can be accumulated in the petals of marigold by selecting a specific combination of isolated DNAs encoding various enzymes involved in the carotenoid biosynthesis pathway to produce antisense RNA, sense RNA or combinations thereof. The present invention also describes isolated DNA sequences encoding the marigold genes beta-cyclase, epsilon-cyclase, beta-hydroxylase, isopentyl pyro-phosphate isomerase.
(2) Description of the Related Art
Carotenoids which comprise the most important group of 40-carbon terpenes and terpenoids are pigments that have a variety of commercial applications. Carotenoids are a class of hydrocarbons (carotenes) and their hydroxylated derivatives (xanthophylls) which comprise 40-carbon (C.sub.40) terpenoids consisting of eight isoprenoid (C.sub.5) units joined together. The terpenoids are joined in such a manner that the arrangement of the isoprenoid units is reversed at the center of the molecule placing the terminal methyl groups in a 1,6 relationship and the non-terminal methyl groups in a 1,5 relationship. Carotenoids can be monocyclic, bicyclic or acyclic. Carotenoids are produced by a wide variety of bacteria, fungi, and green plants. The carotenoids of the most value are intermediates in the carotenoid biosynthetic pathway and consist of lycopene (.psi.,.psi.-carotene), beta-carotene (.beta.,.beta.-carotene), zeaxanthin (.beta.,.beta.-carotene-3,3'-diol), astaxanthin (.beta.,.beta.-carotene-3,3'-diol-4,4'-diketo), lutein (.beta.,.epsilon.-carotene-3,3'-diol) and alpha-carotene (.beta.,.epsilon.-carotene).
Lycopene is a red carotenoid and has utility as a food colorant. Lycopene is naturally synthesized from the precursor phytoene through a series of four separate dehydrogenation steps by the removal of eight atoms of hydrogen. Lycopene is an intermediate in the biosynthesis of other carotenoids in some bacteria, fungi, and all green plants.
Beta-carotene is an orange carotenoid that is naturally produced from lycopene through the intermediate gamma-carotene .beta.,.psi.-carotene) by two sequential cyclization reactions that produce beta rings at the termini. Beta-carotene is useful as a colorant for margarine, butter and cheese, and as a provitamin which has been suggested to have a role in cancer prevention. Current commercial methods for producing beta-carotene include isolation from carrots, chemical synthesis and microbial production.
Zeaxanthin is a yellow carotenoid that is naturally produced from beta-carotene through the intermediate beta-cryptoxanthin by hydrogenation reactions which add hydroxyl groups to the beta rings at both termini. Zeaxanthin is used as a colorant in the poultry industry. Zeaxanthin can be synthesized chemically, however current chemical synthesis reactions are inefficient and are not commercially competitive. Therefore, zeaxanthin is usually produced by extraction from corn grain and corn gluten meal. However, all of these plant sources are characterized by low and inconsistent production levels.
Alpha-carotene is another yellow carotenoid that is naturally produced from lycopene through the intermediate .delta.-carotene (.epsilon.,.psi.-carotene) by two sequential cyclization reactions at the termini that produces one terminus with an epsilon ring and the other terminus with a beta ring. Alpha-carotene is useful as a colorant and as a provitamin.
Carotenoids have a variety of commercial uses ranging from use as a pigment to color foods and cosmetics to uses by the pharmacological industry. Pharmacological uses include use as a control during manufacture to distinguish one drug product from another, as an active component of various medicinal compositions, and as a vitamin supplement for humans. Carotenoids are also used as a dietary supplement in animal and poultry feedstuffs. Carotenoids haven even been used as a photoconductor in recording-media film.
In humans and animals carotenoids have diverse biological functions, and despite the similarity in structure, have different roles. Certain carotenoids are precursors to vitamin A which can be converted to vitamin A by the body, examples are beta-carotene, alpha-carotene, and alpha-cryptoxanthin.
Aside from a role as a precursor to vitamin A, carotenoids are effective quenchers of oxygen free radicals, with lycopene exhibiting the highest quenching activity. Carotenoids function as chain-breaking antioxidants and therefore protect the body from damage by free radicals. Free radicals have been implemented in wide range of human ailments such as onset of pre-mature aging, cancer, atherosclerosis, cataracts, and an array of degenerative diseases. Carotenoids have also been shown to enhance the immune system and to protect the skin from UV damage.
At present only a few plants are widely used to produce carotenoids. However, production of carotenoids from plants is expensive because of the low yields and variability of production. Recombinant DNA technology is a means for increasing the productive capacity of carotenoid biosynthesis in plants.
In U.S. Pat. No. 5,429,939 to Misawa et al DNA segments from Erwinia uredovora encoding bacterial enzymes geranylgeryanyl pyrophosphate synthase, zeaxanthin glycosylase, lycopene cyclase, lycopene synthase, phytoene synthase, and beta-carotene hydroxylase are disclosed. The abovementioned U.S. patent provides a process for producing a carotenoid or a precursor compound in a host but the invention does not provide a means for controlling the ratio of specific carotenoids in a plant.
In U.S. Pat. No. 5,530,188 to Ausich et al DNA segments encoding Erwinia herbicola enzymes geranylgeryanyl pyrophosphate, phytoene synthase, phytoene dehydrogenase-4H, and lycopene cyclase are disclosed. The abovementioned patent provides a means for producing beta-carotene in a plant containing the DNA segment encoding lycopene cyclase. However, the U.S. patent does not provide a means for controlling the ratio of specific carotenoids in a plant thereby producing plants that produce other valuable carotenoids.
In U.S. Pat. No. 5,618,988 to Hauptmann et al, recombinant DNA technology was used to enhance carotenoid accumulation in the storage organs of genetically engineered plants by introducing into the plant a vector comprising a chimeric polypeptide consisting of the bacterial gene encoding phytoene synthase conjugated to a plastid transit peptide. The phytoene synthase was derived from the bacterium Erwinia herbicola. While the abovementioned U.S. patent provides a means for increasing production of phytoene which then serves as a precursor to pigmented carotenoids, the patent does not provide a means for controlling the ratio of specific carotenoids in a plant thereby producing plants that produce specific valuable carotenoids.
In U.S. Pat. No. 5,684,238 to Ausich et al DNA segments from Erwinia herbicola encoding enzymes geranylgeryanyl pyrophosphate synthase, phytoene synthase, phytoene dehydrogenase-4H, lycopene cyclase, beta-carotene hydroxylase, and zeaxanthin glycosylase are disclosed. The abovementioned patent provides a means for producing zeaxanthin or glycosylated zeaxanthin in a culture containing a precursor and a host containing one or more said DNA segments or a transformed plant containing said beta-carotene hydroxylase. However, the U.S. patent does not provide a means for controlling the ratio of other carotenoids in a plant thereby producing plants that produce other valuable carotenoids.
In U.S. Pat. No. 5,744,341 to Cunningham, Jr. et al DNA segments from Arabidopsis thaliana encoding the eucaryote enzymes epsilon-cyclase and beta-hydroxylase, and DNA segments from Arabidopsis thaliana and bacterium Haematococcus pluvialis encoding the enzyme isopentyl pyrophosphate isomerase are disclosed. The U.S. patent suggests uses for the disclosed DNA segments, however the patent does not provide a means for controlling the ratio of specific carotenoids in a plant species using DNA segments encoding various carotenoid biosynthesis enzymes from the same species thereby producing plants that produce other valuable carotenoids.
In U.S. Pat. No. 5,750,865 to Bird et al DNA segments homologous to part or all of the clone pTOM from tomato is provided as a means to modify carotenoid biosynthesis in plants by promoting or inhibiting the synthesis of various carotenoids. The clone pTOM encodes an enzyme with a significant degree of homology to the crtB gene of Rhodobacter capsulatus which encodes phytoene synthase. The abovementioned invention is used to promote or inhibit the carotenoid biosynthetic pathway, but the invention does not provide a means for controlling the ratio of specific carotenoids in a plant.
Although the above techniques have been successful in providing enhanced levels of certain carotenoids in bacterial hosts when the appropriate carotenoid precursor is provided to the host, it would be preferable to utilize a higher plant species wherein technical maintenance procedures would be minimized and yield of specific carotenoids could be optimized. While U.S. patents to Hauptmann et al and Ausich et al disclose uses in higher plants, the carotenoid enzymes disclosed are of bacterial origin which are structurally distinct from the carotenoid enzymes of eucaryote origin. It is well known in the art that an enzyme from a bacterium can be functionally similar to an enzyme from a eucaryote, however the enzymes are rarely structurally related and in many cases the enzymes can possess different secondary functions that in the heterologous host can be undesirable. While U.S. patents to Bird et al and Cunningham et al disclose several DNA segments encoding carotenoid biosynthesis enzymes, the proposed uses for said DNA segments are in heterologous hosts which in certain cases may result in undesirable side effects.
Therefore, there still remains a need for isolation of DNA sequences encoding other carotenoid biosynthetic enzymes from other higher plants. There also remains a need to manipulate the carotenoid biosynthetic pathway in plants to enhance production of specific carotenoid compounds. Finally, there remains a need for transformed plant species, wherein each variety of transformed plant species comprises a combination of DNA sequences derived from a plant which when in the transformed plant species affects the accumulation of specific carotenoid compounds.